In selecting antibiotics for the treatment of bacterial infections, the selection process can vary from relatively straight forward to complex. For example, for streptococcus infections, the antibiotic of choice, penicillin, is well known in the field and can be prescribed with reasonably certainty that it will be effective. For a host of other infections, however, it is not so easily determined what the most efficacious antibiotic or combination of antibiotics will be. This is especially true with resistant strains of bacteria. Typically, the cells of the infecting organism are isolated, purified and then grown in culture in sufficient quantity such that they may be tested against various antibiotics. This is standard procedure. However, the isolation, purification and growth of the cells is normally a time consuming process, say ranging between 12 to 48 hours. In the case of slow growing mycobacteria, the process can be longer.
Common to antibiotic susceptibility tests has been the need to directly or indirectly measure the increase in the mass of a bacterial culture over time. This is accomplished in the presence and absence of antibiotics. Until recently, mass could only be determined conveniently by photometry. Today methods range from measurement of cellular ATP and the release of radioactive carbon dioxide from labelled substrate to release of fluorophores from fluorogenic substrates. Still, all these methods require the growth of the organism and generally need 10.sup.5 -10.sup.7 cells/inoculum. The use of large numbers of cells requires time consuming isolation and growth steps.
The last quarter century, and especially the last decade, has seen a revolution in the application of sensitive and rapid methods of chemical analysis. This has happened, to a large extent, due to advances in electronics, optics, and computer technology which have allowed the practical application of physical methods which previously had been understood in theory, but were too cumbersome to use. These methods have had a major impact on analytical laboratories by making previously difficult analyses affordable and routine by providing many opportunities for automation.
However, until relatively recently, there was little promise of applying these sophisticated new techniques to biodetection because of the lack of information regarding the molecular composition of microorganisms. Today, chemical information can be used effectively to establish relationships at all levels in the taxonomic hierarchy. Chemical properties, it appears, can and must be used in description of many genera and species.
The progress of biochemists and microbiologists in characterizing and identifying chemical markers has not gone unnoticed by chemical analysts. During the past several years, there has been marked progress in methods of chemical analysis and automation in biodetection and identification. Several potentially rapid new physical methods have been developed in the past several years which promise to achieve truly rapid analysis.
Among the most highly developed of the new rapid techniques is mass spectroscopy and its various combinations with gas chromatography (bacterial byproducts from cultures) and pyrolysis methods. Gas chromatography is highly effective in detecting characteristic bacterial metabolic products. Flow cytometry has provided means of the rapid detection, identification, and separation of cells. Total luminescence spectroscopy can detect organisms very rapidly. The various immunological methods also can be very specific and very rapid. All of these methods have their distinct advantages and disadvantages.
Mass spectroscopy may be unequalled in identification of pure cultures, and it is very rapid and sensitive. However, it is expensive to use, requires the destruction of samples, and is of questionable use in the analysis of complex mixtures. Flow cytometry is perhaps even more costly, requires extensive sample preparation, and in many aspects is limited in its scope of applicability. Luminescence techniques are of little use except in studies of pure cultures unless combined with immunological methods. Immunological methods are unequalled in specificity and speed, as well as sensitivity. Yet, they are often impractical to use unless very expensive and perishable materials are available in a state of constant readiness. Such methods are not practical for a wide range of organisms. Gas chromatography requires that cells be grown and, hence, this method is generally slow and of limited applicability.
In U.S. Pat. No. 4,847,198, a system for the rapid detection and identification of bacteria and other microorganisms is disclosed. A beam of visible or ultraviolet light energy contacts a microorganism under investigation. A portion of the light energy is absorbed by the microorganism and a portion of the light energy is `emitted` from the sample at a lower energy level. The emitted light energy (resonance enhanced Raman scattering) may be measured at any angle but preferably is measured as back scattered energy. This energy is processed to produce spectra which are inherently characteristic of the microorganisms.
The light energy which contacts the microorganism can be at any wavelength so long as it corresponds to a molecular electronic transition which corresponds to strong absorption by the organism. Preferably, the energy is a single selected wavelength in the ultraviolet range since most electronic transitions of component molecules of microorganisms occur in that range.
In a preferred embodiment, the emitted energy measured is based upon ultraviolet resonance Raman spectroscopy. Bacteria under investigation are struck by an incident beam of light energy, typically a single wavelength in the ultraviolet range. The emitted energy is collected, collimated and focused onto the entrance slit of a monochromator. The beam strikes a grating or gratings and the wavelengths reflected by the grating or gratings are plotted versus intensity to obtain a spectrum.
The present invention is directed to a previously unrecognized use of the spectra generated according to the teachings of the '198 patent.
The inventive process enormously reduces or eliminates the purification and growth steps of the prior art and also eliminates the need for bacterial mass measurement in the final testing step. The invention utilizes the fact that in the life cycle of a cell exposed to enriched media, there is a phase prior to mitosis recoginized as the lag phase where the RNA accumulates significantly and rapidly. The effect of an antibiotic, rifampin, on this accumulation is used as a measure of its efficacy against the target cell.
The spectra of a first set of target cells as a control are plotted. Target cells then are placed into BHI or other enriched growth medium and divided into second and third sets. In the second set, the cells are cultured under optimal conditions. In the third set, the cells are cultured under the same optimal conditions as the second set but an antibiotic is added to the culture of the second set. Prior to mitosis the cells of the sets are analyzed and the spectra plotted and compared.
In the preferred embodiment of the invention, with E. coli the target cell, the affect on the ribosome peaks are compared to determine the efficacy of the antibiotic. This peak was selected because it reflects the rate of growth of ribosomes which increase in large amounts prior to rapid cell division. It has been found that other lesser peaks in the spectra can also be correlated to ribosomal growth rates and can be used to evaluate the efficacy of the desired antibiotic.